CARM1 hypermethylates the NuRD chromatin remodeling complex to promote cell cycle gene expression and breast cancer development

Abstract Protein arginine methyltransferase CARM1 has been shown to methylate a large number of non-histone proteins, and play important roles in gene transcriptional activation, cell cycle progress, and tumorigenesis. However, the critical substrates through which CARM1 exerts its functions remain to be fully characterized. Here, we reported that CARM1 directly interacts with the GATAD2A/2B subunit in the nucleosome remodeling and deacetylase (NuRD) complex, expanding the activities of NuRD to include protein arginine methylation. CARM1 and NuRD bind and activate a large cohort of genes with implications in cell cycle control to facilitate the G1 to S phase transition. This gene activation process requires CARM1 to hypermethylate GATAD2A/2B at a cluster of arginines, which is critical for the recruitment of the NuRD complex. The clinical significance of this gene activation mechanism is underscored by the high expression of CARM1 and NuRD in breast cancers, and the fact that knockdown CARM1 and NuRD inhibits cancer cell growth in vitro and tumorigenesis in vivo. Targeting CARM1-mediated GATAD2A/2B methylation with CARM1 specific inhibitors potently inhibit breast cancer cell growth in vitro and tumorigenesis in vivo. These findings reveal a gene activation program that requires arginine methylation established by CARM1 on a key chromatin remodeler, and targeting such methylation might represent a promising therapeutic avenue in the clinic.

The NuRD complex (nucleosome remodeling and deacetylase) was first identified as a large multi-subunit complex coupling ATP-dependent chromatin-remodeling and histone deacetylase activities (32)(33)(34)(35).It comprises many different subunits, which include ATP-dependent chromatinremodeling enzymes CHD3 and CHD4, histone chaperones RBBP4 and RBBP7, histone deacetylase HDAC1 and HDAC2, DNA-binding proteins MT A1, MT A2 and MTA3, CpG-binding proteins MBD2 and MBD3, and GA T A zincfinger domain-containing proteins GA T AD2A (p66 α) and GA T AD2B (p66 β) ( 36 ,37 ).More recently, it has been shown that KDM1A (LSD1) is also a component of the NuRD complex, expanding NuRD's chromatin remodeling capacity to include ATPase, histone deacetylase, and histone demethylation activities ( 38 ).The homologs or isoforms of some of the subunits in the NuRD complex were found to be mutually exclusive, leading to a horde of NuRD sub-complexes ( 39 ,40 ).The NuRD complex was initially thought to exclusively repress gene transcription due to its composition, such that HDAC1 / 2 are capable of deacetylating histones and MBDs are connected to DNA methylation ( 32-34 ,41 ).However, recent genomewide data revealed that the NuRD complex can be localized on promoters and enhancers of genes that are actively being transcribed ( 40 ,42-47 ).The exact role of NuRD in regulating gene expression is not clearly understood, but one hypothesis is that it serves to modulate chromatin structure at active transcriptional sites to fine-tune gene expression ( 45 ).Although the change of gene expression by NuRD is moderate, the cumulative effect of this is nevertheless an acute phenotype.Due to its critical roles in regulating basic cellular processes, such as chromatin remodeling, gene transcription, DNA damage repair and cell cycle progression ( 36 , 37 , 48-50 ), alterations of NuRD activity have been shown to lead to developmental defects, cancers, and accelerated ageing (51)(52)(53).
One of the fundamental aspects of understanding the functions of NuRD and the underlying molecular mechanisms is to delineate the NuRD interactome.Efforts have been devoted to investigate the 3D (three dimensional) architecture of the core subunits, aiming to provide insights into the assembly and recruitment of the NuRD complex ( 36 ,37 ).Transcription factors and cofactors, such as GA T A1 / FOG1, SALL1, c-JUN, IKAROS and ZMYND8, have been shown to recruit the NuRD complex to specific gene loci in specific contexts ( 40 ,54-57 ).Inappropriate localization of the NuRD complex has been suggested to contribute to cancer development ( 52 ).Identification of proteins that govern the recruitment of the NuRD complex will shed light on understanding the molecular mechanisms in NuRD-regulated gene transcription.
In the current study, we reported that CARM1 directly interacts the GA T AD2A / 2B subunit in the NuRD complex.They are localized on the promoter regions of and regulate the transcriptional activation of a large cohort of genes with implications in cell cycle control.CARM1-mediated gene activation requires CARM1-mediated GA T AD2A / 2B methylation at a stretch of arginine sites.To underscore the biological significance of CARM1-mediated GA T AD2A / 2B methylation and transcriptional events, high expression of CARM1 and NuRD is observed in clinical breast cancer samples, and knockdown of CARM1 and NuRD significantly attenuates breast cancer cell growth in vitro and tumorigenesis in vivo .

Immunoblotting and immunoprecipitation
Immunoblotting and immunoprecipitation were performed following the protocol described previously ( 59 ,60 ).Antibodies used in the current study was presented in Supplementary Table S3 .

Purification of CARM1-associated proteins
HEK293 cells were transfected with pCMV10-Flag-CARM1-HA, and then selected with G418 (1 mg / ml) (Gibco, 10131035).Resultant single colonies were subjected to immunoblotting to examine the expression of exogenous CARM1 protein.To purify proteins associated with CARM1, cytosolic and nuclear fractions were first separated and nuclear extracts were then prepared in a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA and 1% Triton X-100, followed by affinity purification using Anti-FLAG M2 Affinity gel (Millipore, A2220).DNase I (Promega, M6101) was added during purification to remove genomic DNA.Immunoprecipitates were then washed with low-salt buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA and 1% Triton X-100 for three times followed by high-salt buffer containing 50 mM Tris-HCl (pH 7.4), 420 mM NaCl, 1 mM EDTA and 1% Triton X-100 for two times before elution with 3 × Flag peptides (Sigma, F4799).The eluates were subjected to in solution digestion and LC-MS / MS analysis directly or resolved by SDS-P AGE gel, silver -stained and then subjected to in gel digestion following the protocol described below.
ChIP-seq sample preparation and computational analysis of ChIP-seq data were performed as following.
Library construction: the libraries were constructed following Illumina ChIP-seq Sample prep kit (Illumina, 11257047 A).Briefly, ChIP DNA was end-blunted and added with an 'A' base so the adaptors from Illumina with a 'T' can ligate on the ends.Then 200-400 bp fragments are gel-isolated and purified.The library was amplified by 18 cycles of PCR.
Primary analysis of ChIP-Seq datasets: the image analysis and base calling were performed by using Illumina's Genome Analysis pipeline.The sequencing reads were aligned to hg19 Refseq database by using Bowtie2 ( http://bowtie-bio.sourceforge.net/bowtie2/ index.shtml ) ( 61 ) with default parameters.Both uniquely aligned reads and reads that align to repetitive regions were kept for downstream analysis (if a read was aligned to multiple genomic locations, only one location with the best score was chosen).Clonal amplification was circumvented by allowing maximal one tag for each unique genomic position.The identification of ChIP-seq peaks was performed using HOMER ( http:// biowhat.ucsd.edu/homer ) ( 62 ).The threshold for the number of tags that determined a valid peak was selected at a false discovery rate (FDR) of 0.001.Fourfold more tags relative to the local background region (10 kb) were also required to avoid identifying regions with genomic duplications or non-localized binding.Genomic distribution was done by using the default parameters from HOMER with minor modifications, in which promoter peaks were defined as those with peak center falling between 1000 bp downstream and 5000 bp upstream of transcript start sites (TSSs).Motif analysis was performed using HOMER.Tag density for histograms (50 bp / bin), box plots and heat maps were generated by using HOMER or deep Tools ( https:// deeptools.readthedocs.io/en/ develop ) ( 63 ).Box plots were then generated by R software ( https://www.rproject.org/)and significance was determined using Student's t test.Heat maps were visualized using Java TreeView ( http: //jtreeview.sourceforge.net) ( 64 ) or deep Tools.
ChIP-seq files were deposited in the Gene Expression Omnibus database under accession GSE209910.The following link has been created to allow review of record GSE209910 while it remains in private status: https://www.ncbi.nlm.nih.gov/ geo/ query/ acc.cgi?acc=GSE209910 (token: gzudaioqxxirzwx).
RNA sequencing (RNA-seq) and computational analysis of RNA-seq data Total RNA was isolated using RNeasy Mini Kit (Qiagen, 74104) following the manufacturer's protocol.DNase I (Qiagen, 79254) in column digestion was included to ensure the RNA quality.RNA library preparation was performed by using NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina (Illumina, E7420L).Paired-end sequencing was performed with Illumina HiSeq platform at RiboBio Co., Ltd or Amogene Biotech Co., Ltd.
Sequencing reads were aligned to hg19 Refseq database by using Tophat ( 65 ).Both uniquely aligned reads and the sequencing reads that aligned to repetitive regions were kept for downstream analysis (if a read aligned to multiple genomic locations, only one location with the best score was chosen).Only reads on exons were counted for quantifying gene expression by HOMER.DESeq2 was used to compute the significance of differential expressed genes (FDR ≤ 0.05) ( 66 ).Only coding genes with FPKM larger than 0.5, either in control or siRNA-treated sample, were included in our analysis.FPKM of a gene was calculated as mapped reads on exons divided by exonic length and the total number of mapped reads.Box plots were then generated by R software and significance was determined using Student's t test.Heat maps were visualized using Java TreeView or deepTools.
RNA-seq files were deposited in the Gene Expression Omnibus database under accession GSE209910.The following link has been created to allow review of record GSE209910 while it remains in private status: https://www.ncbi.nlm.nih.gov/ geo/ query/ acc.cgi?acc=GSE209910 (token: gzudaioqxxirzwx).

GST pull-down assay
In vitro purified, bacterially-expressed GST-tagged proteins were incubated with Flag-tagged proteins purified from overexpressed HEK293 cells at 4 • C for at least 4 h.Glutathione agarose (Millipore, G4520) were then washed three times with washing buffer containing 300 mM KCl and 0.05% NP-40 before loading onto SDS-PAGE gel and immunoblotting.

In vitro methylation assay
In vitro methylation assay was performed in methylation buffer (50 mM Tris-HCl, pH 8.0, 20 mM KCl, 5 mM D TT, 4 mM ED TA) in the presence of 1 μCi l -[methyl-3 H]methionine (PerkinElmer, NET061) at 37 • C for 1 h.The reaction was stopped by adding SDS sample buffer followed by SDS-PAGE gel and autoradiogram.

Stable isotope labeling by amino acids in cell culture (SILAC), affinity purification, in solution digestion and LC-MS / MS analysis
Wild-type (WT) and CARM1 knockout (KO) HEK293 cells were grown in SILAC DMEM (Invitrogen, 88364) supplemented with l -lysine / arginine and l -lysine / arginine-U-13C6 (Cambridge Isotope Laboratories, 1119-34-2, 201740-91-2, 657-26-1, 2024-06-540), respectively, together with 10% dialyzed FBS (Gibco, 30067334), l -glutamine (Gibco, A2916801) and penicillin / streptomycin (Gibco, 15140122) for 2 weeks followed by transfected with vectors expressing pCMV10-3xFlag-GA T AD2A for 48 h.Cells were then lysed in a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA and 1% Triton X-100, pooled and subjected to affinity purification by using Anti-FLAG M2 Affinity gel (Millipore, A2220), washed extensively with a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA and 1% Triton X-100, and eluted with 3 × Flag peptides (Sigma, F4799).Eluates were then subjected to in solution digestion following the protocol as described previously ( 20 ).MS experiments were performed on a nanoscale UHPLC system (Thermo Fisher Scientific, EASY-nLC1200) connected to an Orbitrap Fusion Lumos equipped with a nanoelectrospray source (Thermo Fisher Scientific, IQLAAE-GAAPFADBMBHQ).The peptides were separated on a RP-HPLC analytical column (75 μm × 25 cm) packed with 2 μm C18 beads (Thermo Fisher Scientific, 164941) using a linear gradient ranging from 9% to 28% ACN in 90 mins and followed by a linear increase to 45% B in 20 min at a flow rate of 300 nl / min.The Orbitrap Fusion Lumos acquired data in a data-dependent manner alternating between full-scan MS and MS2 scans.The MS spectra (350-1500 m / z ) were collected with 120000 resolution, AGC of 4 × 10 5 , and 50 ms maximal injection time.Selected ions were sequentially fragmented in a 3 s cycle by HCD with 30% normalized collision energy, specified isolated windows 1.6 m / z , 30 000 resolution.AGC of 5 × 10 4 and 120 ms maximal in-jection time were used.Dynamic exclusion was set to 15 s.Raw data was processed using Proteome Discoverer (PD , version 2.1), and MS / MS spectra were searched against the reviewed Swiss-Prot human proteome database.The abundance of peptides was specified by PD 2.1, in which the minimum and maximum value was set to 0 and 200, respectively.For all methylated peptides in GA T AD2A detected, the abundance changed from 200 to 0, indicating that methylation detected on these peptides in WT cells were abolished in CARM1 KO cells.

Mining of the cancer genome atlas (TCGA) data and Kaplan-Meier survival analysis
Expression data (RPKM) of CARM1 and GA T AD2A in a cohort of TCGA clinical breast samples (tumor: 1102; normal: 113) was downloaded from GDC Data Portal.Box plots were generated by R software and significance was determined using Student's t-test.Heat maps were visualized using Java TreeView.Kaplan-Meier survival analyses for OS (overall survival) ( n = 1402) of breast cancer patients were done following the link below: http:// kmplot.com/analysis/ index.php?p=service&default=true .

Cell proliferation assay, FACS (fluorescence-activated cell sorting) analysis, colony formation assay and tumor xenograft assay
Cell viability was measured by using a CellTiter 96 AQueous one solution cell proliferation assay kit (Promega, G3580) following the manufacturer's protocol.Briefly, cells were seeded in culture plates coated with poly-d -lysine (0.1% (w / v), Sigma, P7280) and transfected with siRNA for different time points followed by cell proliferation assay.To measure cell viability, 20 μl of CellTiter 96 AQueous one solution reagent was added per 100 μl of culture medium, and the culture plates were incubated for 1 hr at 37 • C in a humidified, 5% CO 2 atmosphere incubator.The reaction was stopped by adding 25 μl of 10% SDS.Data was recorded at wavelength 490 nm using a Thermo Multiskan MK3 Microplate Reader.
For FACS analysis, cells were trypsinized, washed with PBS and fixed with ethanol at 4 • C overnight.Cells were then washed with PBS and stained with PI / Triton X-100 staining solution (0.1% (v / v) Triton X-100, 0.2 mg / ml DNasefree RNase A (Sigma, R4642), 0.02 mg / ml propidium iodide (Roche, 11348639001)) at 37 • C for 15 min.DNA content was then measured and about 10 5 events were analyzed for each sample.Data were analyzed using ModFit LT (Verity Software House).
For colony formation assays, 2000 cells transfected with siRNA, were maintained in a 6-well plate, and colonies were examined 10 days after.Briefly, colonies were fixed with methanol / acid solution (3:1) for 5 min and stained with 0.1% crystal violet for 15 min.For quantification, the crystal violet dye was released into 10% acetic acid and measured at wavelength 590 nm (OD590).
For tumor xenograft assay, female BALB / C nude mice (age 4-6 weeks) were subcutaneously implanted with 5 × 10 6 of MCF7 cells or MDA-MB-231 cells suspended in DMEM medium without FBS.To promote MCF7 cell tumorigenicity, each nude mouse was brushed with estrogen (E 2 , 10 −2 M) (Sigma, E2758) every 3 days for the duration of the experiments.All mice were euthanized 6 weeks after subcutaneous injection.Tumors were then excised, photographed and weighted.For EZM2302 treatment in vivo , when tumor size reached approximately 100 mm 3 , mice were randomly assigned into three groups and treated with EZM2302 intraperitoneally every 2 days.Tumors were then excised, photographed and weighted.All animals were housed in the Animal Facility at Xiamen University under pathogen-free conditions, following the protocol approved by the Xiamen Animal Care and Use Committee.

CARM1 interacts with the NuRD complex
Our previous study demonstrated that CARM1 methylates a large number of non-histone proteins ( n = 301) with implications in a plethora of cellular processes ( 6 ).To further study the function of CARM1-mediated methylation, we first sought to identify CARM1-associated proteins as which might help us to better define its 'true' substrates.HEK293 cells stably expressing CARM1 were subjected to affinity purification followed by mass spectrometry analysis ( Supplementary Figure S1 A).As previously reported, the SWI / SNF chromatin remodeling complex was found to be abundantly present in proteins associated with CARM1 ( 17 ,67 ) ( Supplementary Figure S1 B).Unexpectedly, all the subunits in a second chromatin remodeling complex, the NuRD complex, were also abundantly present, which included the ATPase CHD3 / 4, histone chaperones RBBP4 / 7, histone deacetylase HDAC1 / 2, DNAbinding proteins MTA1 / 2 / 3, CpG-binding proteins MBD2 / 3, zinc-finger proteins GA T AD2A / 2B and histone demethylase KDM1A (Figure 1 A and B).When we compared CARM1associated proteins ( n = 4805) with CARM1-methylated proteins ( n = 301) we reported previously ( 6 ), there were 199 proteins were found to be in common ( Supplementary Figure S1 C).GA T AD2A / 2B in the NuRD complex was among these 199 proteins.The presence of the entire NuRD complex in the list of CARM1-associated proteins as well as GA T AD2A / 2B in the list of CARM1-methylated proteins caught our attention.
The interaction between CARM1 and each subunit in the NuRD complex or representative subunits in the SWI / SNF complex was confirmed (Figure 1 C and Supplementary Figure S1 D).Similarly, subunits in the NuRD complex were similarly pulled down by CARM1 when we used anti-HA antibody for immunoprecipitation ( Supplementary Figure S1 E).To further support the interaction between CARM1 and NuRD, eluates from GA T AD2A affinity purification exhibited methyltransferase activity towards histone H3, the histone substrate of CARM1 (Figure 1 D).
Next, we examined which subunit in the NuRD complex interacts with CARM1 directly.In vitro GST pull-down assay was performed by mixing purified subunit in the NuRD complex including CHD4 (representing CHD3 / 4), RBBP4 (representing RBBP4 / 7), HDAC1 (representing HDAC1 / 2), MTA1 (representing MTA1 / 2 / 3), MBD3 (representing MBD2 / 3), GA T AD2A (representing GA T AD2A / 2B) or KDM1A with CARM1.The results revealed that CARM1 specifically interacted with GA T AD2A (Figure 1 E, and Supplementary Figure S1 F and G).The interaction between CARM1 and GA T AD2A were also confirmed at endogenous level in HEK293 cells through co-immunoprecipitation analysis (Figure 1 F and G).We then knocked down GATAD2A in HEK293 cells followed by immunoprecipitation with anti-CHD4 antibody to pull down the NuRD complex, and found that the interaction between CARM1 and the NuRD complex was significantly attenuated compared to control cells, supporting the notion that CARM1 interacts with NuRD through GA T AD2A (Figure 1 H).Quantitative MS analysis confirmed that CARM1 association with NuRD complex was attenuated ( Supplementary Figure S1 H).Meanwhile, the integrity of the NuRD complex was jeopardized when GA T AD2A was knocked down ( Supplementary Figure S1 H).
To determine which domain in CARM1 is required for its interaction with GA T AD2A, we transfected HEK293 cells with vectors expressing GA T AD2A and full length (FL), amino (N)-terminal deleted ( N), carboxyl (C)-terminal deleted ( C), or catalytic core only (C.C)-CARM1 followed by immunoprecipitation and immunoblotting.CARM1 Nterminus was found to be required for its interaction with GA T AD2A (Figure 1 I and J).The GA T AD2A-interacting region in CARM1 N-terminus was further narrowed down to amino acid 63-88 (referred as region 3) by GST pull-down assay ( Supplementary Figure S1 I-K).To further confirm that region 3 was required for CARM1 to interact with GA T AD2A, purified full length (FL) or region 3-deleted ( 3) CARM1 was mixed with GA T AD2A or MED12 C-terminus (aa 1616-2177), which was reported to interact with CARM1 ( 5 ).Deletion of region 3 nearly abolished the interaction between CARM1 and GA T AD2A, whereas it had no effects on MED12 C-terminus (Figure 1 K and Supplementary Figure S1 L).Taken together, our data suggested that CARM1 interacts with NuRD through GA T AD2A, and presumably GA T AD2B too, due to high homology.

CARM1 h ypermeth ylates GA T AD2A / 2B in the NuRD complex
Having demonstrated that CARM1 interacts with GA T AD2A / 2B in the NuRD complex, we next sought to fully map the arginine methylation sites in GA T AD2A / 2B in order to understand the function of CARM1-mediated methylation.In our previous report, GA T AD2A and GA T AD2B in the NuRD complex were found to be exclusively methylated by CARM1 (i.e.methylation signals found on all arginine sites in GA T AD2A / 2B were completely abolished when CARM1 was depleted in cells.)( Supplementary Figure S2 A and B) ( 6 ).Due to the high sequence homology between GA T AD2A and GA T AD2B in the region found to be methylated by CARM1, we focused on GA T AD2A only to examine CARM1-mediated methylation.To validate GA T AD2A methylation by CARM1, in vitro methylation assay was performed by mixing individual purified NuRD subunits with recombinant GST-CARM1 proteins.CARM1 was found to predominantly methylate GA T AD2A out of all the subunits tested (Figure 2 A), which was consistent with the observation that GA T AD2A specifically interacts with CARM1 (Figure 1 E).Collaborating with the finding that region 3 in CARM1 was required for CARM1 to target GA T AD2A, deletion of region 3 ( 3) nearly abolished CARM1-mediated GA T AD2A methylation (Figure 2 B).As expected, N-terminal-deleted ( N) and enzymatically dead (M) CARM1 lost their ability to methylate GA T AD2A (Figure 2 B).Serving as a negative control, deletion of region 4 ( 4) exhibited no impact on GA T AD2A methylation (Figure 2 B).Furthermore, deletion of region 3 had no impact on CARM1-mediated methylation on core histones / histone H3 or MED12 ( Supplementary Figure S2 C and D), indicating that although CARM1 utilizes its N-terminus to target multiple substrates, the specific regions mediating these interactions appear to be different.To support that CARM1 methylates GA T AD2A, GA T AD2A methylation was found to be inhibited by EZM2302, a selective inhibitor of CARM1 (Figure 2 C).
To further fully uncover the arginine methylation sites in GA T AD2A and their regulation by CARM1, wild-type or CARM1 knockout cells were subjected to SILAC labeling, transfected with Flag-tagged GA T AD2A, pooled and followed by affinity purification and mass spectrometry analysis (Figure 2 D and Supplementary Figure S2 E).GA T AD2A was found to be hypermethylated at seven arginine sites clustered in the region between the coil-coil domain (CR1) and the C-terminal GA T A-like zinc finger domain (CR2), which were mono-and di-methylated arginine 213 (R213me1 / 2), R225me1, R249me1 / 2, R258me1 / 2, R273me1 / 2, R285me1 and R293me1 / 2 (Figure 2 E, F, Supplementary Figure S2 F and Supplementary Table S1 ).Importantly, the methylation on all arginine sites was abolished in CARM1 knockout cells, further supporting that GA T AD2A was exclusively methylated by CARM1 in cells (Figure 2 F).It should be noted that the composition of the NuRD complex was not significantly altered when CARM1 was depleted based on our mass spectrometry analysis (Figure 2 G).To confirm that GA T AD2A is exclusively methylated by CARM1 in vitro , in vitro methylation assay was performed by mixing purified GA T AD2A with each individual PRMT, PRMT1 to 9. It was found that CARM1 uniquely methylated GA T AD2A (Figure 2 H).The activity of all PRMTs was shown by using core histone as substrates here and in our previous reports ( Supplementary Figure S2 G) ( 58 ).When we mutated all seven arginine sites identified above to lysine in GA T AD2A (GA T AD2A (7R / K)), the methylation by CARM1 was nearly abolished (Figure 2 I).Taken together, GA T AD2A in the NuRD complex was specifically and exclusively methylated by CARM1 at a cluster of arginine sites.

CARM1 and NuRD complex occupy a large number of chromatin sites in common
The observation that CARM1 interacts and methylates the NuRD complex prompted us to examine the biological significance of this connection between CARM1 and the NuRD complex.Both CARM1 and NuRD have been reported to bind to chromatin to regulate gene transcription.We first tested whether CARM1 and NuRD co-localize on chromatin.ChIP-seq for CARM1 and representative subunits in NuRD including CHD4, HDAC2, GA T AD2A and KDM1A was performed in HEK293 cells.A large number of CARM1 binding sites were detected, the majority of which were found to be localized at gene promoter regions (Figure 3 A).We then compared the binding sites of CHD4, HDAC2, GA T AD2A and KDM1A detected from ChIP-seq to that of CARM1.Tag density plot and heat map demonstrated that CHD4, HDAC2, GA T AD2A and KDM1A largely bound to the same genomic region as CARM1 (Figure 3 B and C).ChIP-seq tag density of CHD4, HDAC2, GA T AD2A and KDM1A was highly correlated to that of CARM1 (Figure 3 D-G and Supplementary Figure S3 A-D).Binding of CARM1, CHD4, HDAC2, GA T AD2A and KDM1A detected from ChIP-seq on representative genes was shown as indicated (Figure 3 H, I and Supplementary Figure S3 E-H).Taken together, CARM1 and  NuRD occupy a large number of promoter sites on chromatin in common.

CARM1 and NuRD activate a large set of cell cycle genes to promote cell cycle progression in a CARM1 enzymatic activity-dependent manner
The fact that CARM1 and NuRD bind to a large set of gene promoter regions in common prompted us to examine whether they regulate the expression of these genes.To this end, HEK293 cells were transfected with control siRNA or siRNA specifically targeting CARM1 or representative subunits in the NuRD complex including CHD4 , HDAC2 , GATAD2A and KDM1A followed by RNA-seq analysis.To support the notion that CARM1 and NuRD largely localize on the same regions on chromatin, the impact of knockdown of CHD4, HDAC2, GA T AD2A and KDM1A on gene expression was well correlated with that of CARM1 ( Supplementary Figure S4 A-D).In total, 441 genes were significantly and positively regulated (FC ≥ 1.2) by CARM1 and NuRD in common, of which around 70% had CARM1 binding on their promoters, suggesting that CARM1 largely regulated these genes directly (Figure 4 A).In contrast, for genes negativelyregulated by CARM1 and NuRD in common, only around 30% of them exhibited CARM1 and NuRD binding on promoter regions, which was close to background considering the large number of CARM1-bound promoter sites ( n = 8250) detected in the genome.The impact of knockdown of CARM1, CHD4, HDAC2, GA T AD2A and KDM1A on the expression of these 441 genes positively-regulated by CARM1 was shown by heat map and box plot (Figure 4 B  and C).Importantly, for these CARM1 and NuRD commonly regulated genes, cell cycle was one of the most enriched terms when we performed gene ontology (GO) analysis ( Supplementary Figure S4 E).Regulation of cell cycle genes by CARM1, CHD4, HDAC2, GA T AD2A and KDM1A was validated by RT-qPCR analysis for representative genes (Figure 4 D).We then tested whether CARM1 and NuRD regulate cell cycle progression.HEK293 cells were transfected with control siRNA or siRNA specifically targeting CARM1 , CHD4 , HDAC2 , GATAD2A or KDM1A followed by cell proliferation measurement and FACS analysis.Cell proliferation rate was decreased significantly and number of cells in G1 was increased when knocking down CARM1 or each of these subunits in the NuRD complex (Figure 4 E and F).
The transcriptional activation of representative cell cycle genes appeared to be associated with CARM1's enzymatic activity as wild-type CARM1 (CARM1 (WT)) partially rescued the effects of CARM1 knockdown, but its enzymatically deficient mutant (CARM1 (M)) did so in a much less extent (Figure 4 G).Accordingly, regulation of cell cycle progression by CARM1 was dependent on its enzymatic activity (Figure 4 H).CARM1 (WT) and (M) were expressed equally well (Figure 4 I).Enzymatic activity-dependency to regulate transcription and cell cycle progression by CARM1 was also demonstrated by doing rescue experiments in CARM1-knockout (KO) cells ( Supplementary Figure S4 F-H).The expression of representative cell cycle genes, cell proliferation, and cell cycle progression were inhibited by EZM2302, further supporting that the regulation of these events by CARM1 is dependent on its enzymatic activity (Figure 4 J-L).Taken together, our data revealed that CARM1 and NuRD regulate a large cohort of genes to control cell cycle progression.

CARM1-mediated GA T AD2A methylation is involved in NuRD chromatin binding, transcriptional activation of cell cycle genes, and cell cycle progression
Enzymatic activity-dependency of CARM1 in the transcriptional activation of cell cycle genes and cell cycle progression suggested that CARM1-mediated GA T AD2A methylation might be functionally important.Firstly, we tested whether CARM1-mediated methylation is involved in GA T AD2A and therefore NuRD binding on chromatin.ChIP-seq analysis of GA T AD2A was performed in both WT and CARM1 (KO) HEK293 cells.The results showed that GA T AD2A binding was significantly attenuated on genes regulated by CARM1 and NuRD in common in CARM1 KO cells (Figure 5 A and B).GA T AD2A binding in WT or CARM1 KO cells was shown on representative genes (Figure 5 C, D and Supplementary Figure S5 A-C).The impact of knockdown of CARM1 on GA T AD2A binding was further validated by ChIP-qPCR analysis for representative genes (Figure 5 E).CARM1 effects on GA T AD2A chromatin binding appeared to be largely dependent on CARM1's enzymatic activity as CARM1 (WT) largely rescued GA T AD2A binding on chromatin, while CARM1 (M) did so in a much less extent ( Supplementary Figure S5 D).We also examined the binding of other subunits in the NuRD complex, exemplified by HDAC2 and KDM1A, in response to CARM1 depletion, and found that binding of HDAC2 and KDM1A was also significantly reduced ( Supplementary Figure S5 E-H).Collaborating with the observation that GA T AD2A was required for the integrity of NuRD complex, knockdown of GA T AD2A led to significantly decreased binding of CARM1 ( Supplementary Figure S5 I and J).Also, the binding of another subunit of NuRD, CHD4, was significantly decreased when GA T AD2A was knocked down ( Supplementary Figure S5 K).Secondly, ChIP-seq analysis of GA T AD2A (WT) and 7R / K mutant was performed, and the binding of 7R / K mutant was found to be significantly lower on genes co-regulated by CARM1 and NuRD compared to GA T AD2A (WT) (Figure 5 F and G).Binding of GA T AD2A (WT) and 7R / K mutant was shown on representative genes (Figure 5 H, I and Supplementary Figure S5 L-N), and further validated by ChIP-qPCR analysis (Figure 5 J).
We next examined whether CARM1-mediated methylation on GA T AD2A is involved in the transcriptional activation of CARM1 and NuRD commonly regulated genes and cell cycle progression.HEK293 cells were transfected with control siRNA or siRNA specifically targeting GATAD2A in the presence or absence of control vector or vector expressing GA T AD2A (WT) or 7R / K mutant, followed by gene expression and cell cycle analysis.It was found that, compared to GA T AD2A (WT), the 7R / K mutant was much less efficient to rescue the gene expression defect and cell cycle arrest caused by GA T AD2A knockdown (Figure 5 K and L).GA T AD2A (WT) and 7R / K mutant expressed equally well as examined by both RT-qPCR and immunoblotting analysis (Figure 5 M and Supplementary Figure S5 O).It should be noted that the 7R / K mutant appeared to have no impact on the integrity of the NuRD complex compared to GA T AD2A (WT), which was consistent with our observation that CARM1 exhibited no significant impact on the integrity of the NuRD complex ( Supplementary Figure S5 P).Taken together, our data suggested that CARM1-mediated methylation is involved in the chromatin binding of the NuRD complex, the transcriptional activation of cell cycle genes, and cell cycle progression.WT and GA T AD2A mutant (7R / K) ChIP-seq on E2F8 (H) and CDC25A (I) gene are shown.( J ) HEK293 cells as described in (F) were subjected to ChIP-qPCR analysis with primers specifically targeting the promoter region of selected cell cycle genes as indicated ( ± s.e.m., ** P < 0.01, *** P < 0.001).( K ) HEK293 cells were transfected with control siRNA or siRNA targeting 3 UTR region of GA T AD2A together with or without control vector or vectors expressing HA-tagged WT or mutant GA T AD2A (7R / K), followed by RT-qPCR analysis to examine the expression of selected cell cycle genes as indicated, and data was represented by heat map.( L ) HEK293 cells as described in (K) were subjected to FACS analysis.( M ) HEK293 cells as described in (K) were subjected to RT-qPCR analysis to examine the expression of GA T AD2A.

CARM1-mediated GA T AD2A methylation is required for breast cancer cell growth both in vitro and in vivo
The functional role of CARM1-mediated GA T AD2A methylation in cell cycle gene transcriptional activation and cell cycle progression prompted us to examine its role in cancer.We focused on breast cancer in the current study as numerous studies including ours showed that CARM1 plays a critical role in breast cancer development ( 5 ,68 ).Both CARM1 and GA T AD2A were found to be expressed higher in breast tumor than normal samples, and their high expression predict poor prognosis in breast cancer patients ( Supplementary Figure S6 A-D).The direct interaction between CARM1 and GA T AD2A was verified in MCF7 cells with co-immunoprecipitation analysis (Figure 6 A and B).We then performed ChIP-seq for both CARM1 and GA T AD2A in MCF7 cells.Tag density plot and heat map demonstrated that both proteins were largely co-localized in the genome ( Supplementary Figure S6 E and F).ChIP-seq tag density of CARM1 was highly correlated to that of GA T AD2A ( Supplementary Figure S6 G and H).Binding of CARM1and GA T AD2A detected from ChIP-seq on representative genes was shown ( Supplementary Figure S6 I-K).Knockdown of CARM1 led to a significant reduction of GA T AD2A methylation as well as GA T AD2A binding on the promoter regions of representative cell cycle genes (Figure 6 C and D).Consequently, knockdown of CARM1 and GA T AD2A resulted in a decreased expression of these cell cycle genes (Figure 6 E).
Next, we examined whether CARM1-madiated GA T AD2A methylation is required for breast cancer cell growth and tumorigenesis.Cell proliferation rate was decreased significantly and cells were arrested at G1 phase when CARM1 or GA T AD2A was knocked down in MCF7 cells (Figure 6 F and G).The effects on MCF7 cell growth were further demonstrated by colony formation assay (Figure 6 H and I).Knockdown efficiency of siCARM1 and siGA T AD2A was demonstrated by RT-qPCR analysis ( Supplementary Figure S6 L).The effects of CARM1 and GA T AD2A on cell proliferation, cell cycle progression, and colony formation were also demonstrated in MDA-MB-231 cells ( Supplementary Figure S6 M-P).We then tested whether CARM1-mediated GATAD2A methylation is involved in cell proliferation of MCF7 cells by transfecting cells with control shRNA or shRNA specifically targeting GATAD2A in the presence or absence of control vector or vector expressing GA T AD2A (WT) or 7R / K mutant, followed by cell proliferation analysis.The 7R / K mutant was less efficient to rescue the defects in cell proliferation caused by GA T AD2A knockdown (Figure 6 J).We further tested the effect of CARM1 and GA T AD2A on tumor growth in vivo by injecting nude mice subcutaneously with control MCF7 cells or cells infected with shRNA targeting CARM1 or GATAD2A , and then treated with estrogen to stimulate tumor growth.Knockdown of CARM1 and GA T AD2A significantly attenuated estrogen-induced tumorigenesis (Figure 6 K).Knockdown efficiency of shCARM1 and shGA T AD2A was demonstrated by RT-qPCR analysis ( Supplementary Figure S6 Q).The involvement of CARM1mediated GA T AD2A methylation in tumor growth was also tested.In consistent with what we observed in cultured cells, GA T AD2A 7R / K mutant was less effective in rescuing the tumor growth defects caused by GA T AD2A knockdown (Figure 6 L).GA T AD2A (WT) and 7R / K mutant were expressed equally well ( Supplementary Figure S6 R).Taken to-gether, our data suggested that CARM1-mediated GA T AD2A methylation is involved in breast cancer cell growth and tumorigenesis.

Targeting CARM1 with CARM1 inhibitors inhibits breast cancer cell growth both in vitro and in vivo
The functional role of CARM1-mediated GA T AD2A methylation in breast cancer cell growth prompted us to examine whether targeting CARM1-mediated GA T AD2A methylation using CARM1 inhibitors, EZM2302 and TP-064, could inhibit breast cancer cell growth.MCF7 cells infected with lentivirus expressing GA T AD2A were treated with EZM2302 followed by immunoprecipitation and immunoblotting to detect GA T AD2A methylation.EZM2302 treatment led to a significant reduction of GA T AD2A methylation (Figure 7 A).Consequently, GA T AD2A binding on the promoter regions on representative cell cycle genes and the expression of these cell cycle genes were significantly attenuated in the presence of EZM2302 (Figure 7 B and C).Furthermore, cell proliferation rate was decreased significantly and cells were arrested at G1 phase when MCF7 cells were treated with EZM2302 (Figure 7 D and E).The effects of EZM2302 on MCF7 cell growth were also demonstrated by colony formation assay (Figure 7 F and G).Another inhibitor of CARM1, TP-064, was also found to be effective in inhibiting MCF7 cell proliferation, cell cycle progression, and colony formation ( Supplementary Figure S7 A-D) ( 69 ).Furthermore, EZM2302 significantly attenuated the weight and growth rate of MCF7 cells-derived tumors in mice (Figure 7 H-J), while it displayed minimal effects on the body weight of mice ( Supplementary Figure S7 E).To link CARM1-regulated cell cycle gene transcription to EZM2302 effects on tumor growth, the expression of cell cycle genes including CDK4, CDC25A and CDC25B was significantly inhibited by EZM2302 treatment (Figure 7 K).
To further strengthen the functional role of CARM1mediated GA T AD2A methylation in breast cancer, EZM2302 significantly inhibited GA T AD2A methylation and GA T AD2A binding on the promoter regions of representative cell cycle genes in MDA-MB-231 cells ( Supplementary S7 F and G).The expression of these genes was reduced with EZM2302 treatment ( Supplementary Figure S7 H).Consequently, cell proliferation, cell cycle progression, and formation were significantly inhibited when MDA-MB-231 were treated with EZM2302 ( Supplementary Figure S7 I-L).Furthermore, EZM2302 significantly the weight and growth of MDA-MB-231 cells-derived tumors, while exhibited no significant impact on body weight ( Supplementary S7 M-P).The expression representative cell cycle genes was found to be significantly inhibited by in tumor samples ( Supplementary Figure S7 Q).Taken together, our data suggested that targeting CARM1 is effective in inhibiting breast cancer cell growth both in vitro and in vivo .

Discussion
CARM1 was identified to contain histone arginine methyltransferase and serves as a transcriptional tor .However , the substrates utilized by CARM1 orchestrate transcriptional regulation remain incompletely understood.In the current study, we found that CARM1 and NuRD interact, occupy a large number chromatin sites in common, commonly regulate the transcriptional activation of a   set of cell cycle genes, and promote cell cycle progression.CARM1 methylates one of the subunits, GA T AD2A, in NuRD to regulate its binding with chromatin and function in gene transcription and cell cycle control.The clinical relevance of CARM1-mediated GA T AD2A methylation in cell cycle control is further illustrated in breast cancer, where higher expression of CARM1 and GA T AD2A is observed compared to normal tissues.Knockdown of CARM1 and GA T AD2A significantly attenuates breast cancer cell growth both in vitro and in vivo .Targeting CARM1 with EZM2302, a CARM1-specific inhibitor, significantly inhibits CARM1-mediated GA T AD2A methylation, cell cycle gene transcription, cell cycle progression, and breast tumor growth (Figure 8 ).
Despite that CARM1 has been shown to interact with a plethora of proteins and have a large set of substrates, a deep characterization of its interactome is still needed to fully understand its diverse functions in both physiological and pathological conditions.Through affinity purification and mass spectrometry analysis, we demonstrated that, in addition to the SWI / SNF, CARM1 also interacts with another chromatin remodeling complex, the NuRD complex.The observed interaction between CARM1 and NuRD is intriguing to us for several reasons.Firstly, despite that NuRD was initially thought to exclusively repress gene transcription, genomewide mapping experiments suggested that NuRD, including CHD3 / Mi-2a, CHD4 / Mi-2b, GA T AD2A and GA T AD2B sub-complexes, might be tightly linked to transcriptional activation.The observed interaction between CARM1 and NuRD suggests that CARM1 might provide a molecular basis for NuRD in gene activation.Secondly, both CARM1 and NuRD have been shown to control cell cycle progression, but whether they cooperate to do so and the underlying molecular mechanisms remain elusive.Finally, during the course of mapping CARM1 substrates in the proteome, the methylation of GA T AD2A / 2B subunit in the NuRD complex was found to be dependent on CARM1, suggesting a functional connection between CARM1 and the NuRD complex ( 6 ).It should also be noted that, though CHD3 / Mi-2a and CHD4 / Mi-2b or GA T AD2A and GA T AD2B sub-complexes were shown to be mutually exclusive, both were found to interact with CARM1, suggesting that CARM1 might be a common molecule utilized by all sub-complexes to regulate gene expression.CARM1 specifically and directly interacts with GA T AD2A / 2B through β-strands in the N-terminus (aa 63-88), which appeared to be different from the interacting region that CARM1 utilizes to target other substrates, such as MED12 and histone H3.Interestingly, several other complexes were also identified to be associated with CARM1 including the DNA replication factor C complex, the THO complex, the Prp19 complex, the TFIIIC complex, among others, suggesting CARM1 might also play important roles in cellular processes such as DNA replication, RNA splicing, and tRNA transcription.
The connection between CARM1 and NuRD was further demonstrated on chromatin.The co-localization of CARM1 and NuRD on chromatin prompted us to examine whether they regulate gene transcription in a similar fashion.Knockdown of CARM1 or NuRD resulted in a moderate but significant and reproducible effect on a large cohort of genes.Among all the functions related to the genes regulated by CARM1 and NuRD, cell cycle regulation was one of the most prevalent.Indeed, the cumulative effects of CARM1 and NuRD coregulated transcription were essential for cell cycle progression.The NuRD complex was shown to be recruited to and function locally on DNA damage sites ( 50 ,70 ).In our transcriptomics analysis herein, we noted that a cohort of p53 target genes with implications in DNA damage response were also regulated by CARM1 and NuRD, suggesting they might function in DNA damage response through regulating gene transcription, which adds another layer of complexity to the means of NuRD function in DNA damage response.CARM1 and NuRD complex's function in gene transcriptional regulation in response to DNA damage signals as well as the connection with p53 protein is worthy of future investigation.
In addition of methylating histones to activate gene transcription, CARM1 also methylated the GA T AD2A subunit in the NuRD complex to control its recruitment onto chromatin.A unique feature of CARM1-mediated arginine methylation was that the arginine residues being methylated form a cluster, exhibiting a hypermethylation status, such that there were seven arginine residues in total found to be methylated in a window < 100 amino acids in GA T AD2A.This type of hypermethylation appeared to be a common strategy utilized by CARM1 to target its substrates, such as MED12 and Notch1 as previously reported ( 9 ,20 ), and many others identified in our experiments to globally map CARM1 substrates ( 5 ,6 ).Notably, H4R3me2a, H3R17me2a and methylated arginine 1810 (R1810) in the C-terminal domain (CTD) of RNA polymerase II (RNA Pol II) mediated by CARM1 were shown to be recognized by the tudor-domain of TDRD3, which functions as a transcriptional coactivator ( 21 ,71 ).Therefore, we propose that GA T AD2A methylation might recruit arginine methylation readers such as TDRD3 to activate genes involved in cell cycle regulation.We propose that this type of hypermethylation might serve as 'amplifier' to ensure the recruitment of methylation reader proteins to activate gene transcription.
Due to the fact that they are highly expressed in breast tumor samples and their high expression predict poor prognosis in patients with breast cancer, the pathological relevance of CARM1 and NuRD was investigated.We demonstrated that CARM1-mediated GA T AD2A methylation was important for the growth of breast cancer cells both in vitro and in vivo .CARM1's function in breast cancer is, at least partially, mediated through its methyltransferase activity targeting GA T AD2A.Our data thereby suggested that CARM1-mediated GA T AD2A methylation might serve as a potential druggable target in breast cancer.Indeed, EZM2302, a CARM1-specific inhibitor, inhibits CARM1mediated GA T AD2A methylation, GA T AD2A chromatin binding, transcriptional activation of cell cycle genes, cell cycle progression, and eventually tumor growth.Therefore, targeting CARM1 enzymatic activity or peptide mimics interfering CARM1-mediated GA T AD2A / 2B methylation will provide a new therapeutic avenue for treating breast cancer as well as other CARM1-dependent cancers.

Figure 1 .
Figure 1.CARM1 interacts with the NuRD complex.( A ) HEK293 cells stably expressing Flag-HA-tagged CARM1 or empty vector were subjected to affinity purification, and the eluates were resolved by SDS-PAGE gel, silver-stained and subjected to mass spectrometry analysis.( B ) Subunits in the NuRD complex and the corresponding number of unique peptides identified are shown as indicated.( C ) HEK293 cells as described in (A) were subjected to immunoprecipitation (IP) with anti-Flag antibody f ollo w ed b y immunoblotting (IB) with antibodies as indicated.( D ) In vitro methylation assay w as perf ormed b y mixing core histones with eluates from control v ector or Flag-tagged G A T AD2A affinity purification in HEK293 cells, f ollo w ed b y autoradiogram (upper panel).Loading of core histones is shown by coomassie blue staining (CBS) (bottom panel).( E ) GST pull-down assay was perf ormed b y mixing Flag-tagged G A T AD2A purified from o v er-e xpressed HEK293 cells with GS T or GS T-tagged CARM1 f ollo w ed b y immunoblotting (IB) with anti-Flag antibody.( F, G ) HEK293 cells were subjected to co-immunoprecipitation (Co-IP) assay with control IgG, anti-GA T AD2A (F), or anti-CARM1 (G) antibody, f ollo w ed b y immunoblotting (IB) analy sis with antibodies as indicated.( H ) HEK293 cells transfected with control siRNA (siCTL) or siRNA specifically targeting GA T AD2A (siGA T AD2A) were subjected to immunoprecipitation (IP) with anti-CHD4 antibody, followed by immunoblotting (IB) with antibodies as indicated.( I ) Schematic representation of full length (FL) and truncated CARM1 proteins.N: amino (N)-terminus deletion; C: carboxyl (C)-terminus deletion; C.C: catalytic core.( J ) HEK293 cells were transfected with vectors expressing Flag and HA-tagged GA T AD2A and empty vector, full length (FL), amino (N)-terminal deletion ( N), carboxyl (C)-terminal deletion ( C) or catalytic core only (C.C) CARM1 followed by immunoprecipitation (IP) with anti-HA antibody and immunoblotting (IB) with anti-Flag or anti-HA antibody.( K ) HEK293 cells were transfected with vectors expressing HA-tagged GA T AD2A and empty vector , full length (FL), or region 3-deleted ( 3) CARM1 f ollo w ed b y immunoprecipitation (IP) with anti-HA antibody and immunoblotting (IB) with anti-Flag or anti-HA antibody.

Figure 2 .
Figure 2. CARM1 h ypermeth ylates G A T AD2A / 2B in the NuRD comple x. ( A ) In vitro meth ylation assa y w as perf ormed b y mixing GS T-tagged CARM1 purified from bacterial cells with Flag-tagged, representative subunits in the NuRD complex including RBBP4, RBBP7, HD A C1, HD A C2, MT A1, MT A2 (truncation), MBD3, GA T AD2A and KDM1A purified from over-expressed HEK293 cells, followed by autoradiogram to examine CARM1-mediated methylation (upper panel) or immunoblotting (IB) using anti-Flag antibody to examine the expression of the NuRD subunits (bottom panel).Black arrow and bracket (upper panel) indicated methylation of GA T AD2A and its proteolytic fragments, respectively.White arrows indicated the size of the corresponding subunits in the NuRD complex.( B ) In vitro methylation assay was performed by mixing purified GA T AD2A with full length (FL), region 3-deleted ( 3), region 4 deleted-( 4), amino (N)-terminal-deleted ( N), or enzymatic dead (M) CARM1, f ollo w ed b y immunoblotting (IB) with antibodies as indicated.( C ) HEK293 cells were transfected with or without Flag-tagged GA T AD2A and treated with or without EZM2302 (50 μM, 48 h), f ollo w ed b y immunoprecipitation (IP) with anti-Flag antibody and immunoblotting (IB) analysis with antibodies as indicated.( D ) Schematic representation of the protocol applied for detecting differential binding proteins and post-translational modifications (PTMs) of GA T AD2A in WT and CARM1 (KO) HEK293 cells.WT and CARM1 (KO) HEK293 cells were subjected to SILAC labeling and then transfected with vectors expressing Flag-tagged GA T AD2A.Cells were then lysed, pooled and subjected to affinity purification using M2 agarose followed by mass spectrometry (MS) analysis.( E ) Schematic representation of domain architecture of GA T AD2A.Arginine methylation sites are shown by matc hstic ks.CR: Coil-coil region; FL: full-length.( F ) Arginine methylation sites identified in GA T AD2A through mass spectrometry analysis as shown in (C).me1: mono-methylation; me2: di-methylation.( G ) List of subunits in the CARM1 and NuRD complex identified to be associated with GA T AD2A by mass spectrometry (MS) analysis, and the number of peptides and the ratio of the abundance of each subunit in WT and CARM1 (KO) cells are shown.( H ) In vitro methylation assay was performed by mixing Flag-tagged GA T AD2A with PRMT proteins purified from o v er-e xpressed HEK293 cells as indicated, f ollo w ed b y autoradiogram (upper panel) or immunoblotting (IB) using anti-Flag antibody (bottom panel).White arrows indicated the expression of all PRMTs.Star indicated methylation of GA T AD2A.( I ) In vitro methylation assay was performed by mixing GST-tagged CARM1 purified from bacterial cells with Flag-tagged wild-type (WT) or GA T AD2A mutant with seven arginine methylation sites substituted with lysines (7R / K) purified from over-expressed HEK293 cells, followed by autoradiogram (upper panel) or immunoblotting (IB) using anti-Flag antibody (bottom panel).

Figure 3 .
Figure 3. CARM1 and NuRD complex occupy a large number of chromatin sites in common.( A ) Genomic distribution of CARM1 binding sites identified by ChIP-seq.( B, C ) Histogram (B) and heat map (C) representation of CARM1, CHD4, HD A C2, GA T AD2A and KDM1A ChIP-seq tag density centered on CARM1 binding sites.bp: base pair.( D-G ) B o x plot representation of ChIP tag density (log 2 ) of CHD4 (D), HD A C2 (E), KDM1A (F) and GA T AD2A (G) on CARM1 binding sites, which were divided into three sub-classes, high, medium and low based on ChIP-seq tag density ( ± s.e.m., *** P < 0.001).( H, I ) Genome browser views of CARM1, CHD4, HD A C2, GA T AD2A and KDM1A ChIP-seq on E2F8 (H) and CDC25A (I) genes.

Figure 4 .
Figure 4. CARM1 and NuRD activate a large set of cell cycle genes to promote cell cycle progression in a CARM1 enzymatic activity-dependent manner.( A ) HEK293 cells were transfected with control siRNA ( siCTL ) or siRNA specific against CARM1 (si CARM1 ) or representative subunits in NuRD including CHD4 , HD A C2 , GA T AD2A and KDM1A (si CHD4 , si HD A C2 , si GA T AD2A and si KDM1A ) f ollo w ed b y RNA-seq analy sis.Genes positiv ely -regulated b y CARM1, CHD4, HD A C2, GA T AD2A, and KDM1A in common are sho wn b y v enn diagram ( q < 0.05).( B , C ) T he e xpression le v els (FPKM, log 2 ) for those 441 genes as described in (A) are shown by heat map (B) and box plot (C).( D ) HEK293 cells as described in (A) were subjected to R T-qPCR analy sis to e xamine the e xpression of selected cell cy cle genes as indicated, and data w as represented b y heat map.( E, F ) HEK293 cells as described in (A) were subjected to cell proliferation assay (E) and FACS analysis (F). ( G-I ) HEK293 cells were transfected with control siRNA ( siCTL ) or siRNA specifically targeting CARM1 (si CARM1 ) in the presence or absence of control vector or vector expressing wild-type CARM1 (WT) or its enzymatically deficient mutant (M), f ollo w ed b y R T-qPCR analy sis to e xamine the e xpression of selected cell cy cle genes as indicated (G) and CARM1 (I), and FACS analysis to c hec k cell cycle progression (H).( J ) HEK293 cells treated with or without EZM2302 (50 μM, 48 h) were subjected to RNA extraction and RT-qPCR analysis to examine the expression of selected cell cycle genes as indicated.( K, L ) HEK293 cells treated with or without EZM2302 at concentration as indicated were subjected to cell proliferation assay (K) and FACS analysis (L) ( ± s.e.m., ** P < 0.01, *** P < 0.001).

Figure 5 .
Figure 5. CARM1-mediated h ypermeth ylation of GATAD2A is in v olv ed in NuRD chromatin binding, transcriptional activation of cell cycle genes, and cell cycle progression.( A, B ) GA T AD2A ChIP-seq was performed in WT or CARM1 (KO) HEK293 cells, and heat map (A) and box plot (B) representation of GA T AD2A ChIP-seq t ag densit y centered on transcription st art sites (TSSs) of genes positiv ely -regulated b y CARM1 and NuRD is shown.( C, D ) Genome bro wser vie ws of CARM1 and G A T AD2A ChIP -seq, either in WT or CARM1 (K O) HEK293 cells, on E2F8 (C) and CDC25A (D) gene is shown.( E ) WT or CARM1 (KO) HEK293 cells were subjected to ChIP with anti-IgG or anti-GA T AD2A specific antibody followed by qPCR analysis with primers specifically targeting promoter regions of selected cell cycle genes as indicated ( ± s.e.m., * P < 0.05, *** P < 0.001).( F, G ) HEK293 cells were transfected with siRNA targeting 3 UTR region of GA T AD2A together with or without control vector or vectors expressing HA-tagged WT or mutant GA T AD2A (7R / K), f ollo w ed b y ChIP-seq with anti-HA antibody.Heat map (F) and bo x plot (G) represent ation of ChIP-seq t ag densit y of WT or mut ant GA T AD2A (7R / K) HEK293 cells centered on transcription start sites (TSSs) of genes positiv ely -regulated b y CARM1 and NuRD.( H, I ) Genome browser views of GA T AD2A WT and GA T AD2A mutant (7R / K) ChIP-seq on E2F8 (H) and CDC25A (I) gene are shown.(J ) HEK293 cells as described in (F) were subjected to ChIP-qPCR analysis with primers specifically targeting the promoter region of selected cell cycle genes as indicated ( ± s.e.m., ** P < 0.01, *** P < 0.001).( K ) HEK293 cells were transfected with control siRNA or siRNA targeting 3 UTR region of GA T AD2A together with or without control vector or vectors expressing HA-tagged WT or mutant GA T AD2A (7R / K), followed by RT-qPCR analysis to examine the expression of selected cell cycle genes as indicated, and data was represented by heat map.( L ) HEK293 cells as described in (K) were subjected to FACS analysis.( M ) HEK293 cells as described in (K) were subjected to RT-qPCR analysis to examine the expression of GA T AD2A.

Figure 6 .
Figure 6.CARM1-mediated GATAD2A methylation is required for breast cancer cell growth both in vitro and in vivo .(A, B) MCF7 cells were subjected to co-immunoprecipitation (Co-IP) assay with control IgG, anti-GA T AD2A (A), or anti-CARM1 (B) antibody and followed by immunoblotting (IB) with antibodies as indicated.( C ) MCF7 cells were transfected with siRNA ( siCTL ) or siRNA specific against CARM1 (si CARM1 ) and then infected with lenti-viral vector expressing Flag-tagged GA T AD2A, followed by immunoprecipitation (IP) with anti-Flag antibody and immunoblotting (IB) analysis with antibodies as indicated.( D ) MCF7 cells transfected with siRNA ( siCTL ) or siRNA specific against CARM1 (si CARM1 ) were subjected to ChIP with control IgG or anti-GA T AD2A specific antibody, followed by qPCR analysis with primers specifically targeting promoter regions of selected cell cycle genes as indicated ( ± s.e.m., *** P < 0.001).( E-H ) MCF7 cells transfected with control siRNA ( siCTL ) or siRNA specific against CARM1 (si CARM1 ) or GATAD2A (si GA T AD2A ) were subjected to RNA extraction and RT-qPCR analysis to examine the expression of selected cell cycle genes as indicated (E), cell proliferation assay (F), FACS analysis (G), and colony formation assay (H) ( ± s.e.m., *** P < 0.001).( I ) Quantification of the crystal violet dye as shown in (H).( J ) MCF7 cells were infected with control shRNA (shCTL) or shRNA targeting 3 UTR region of GA T AD2A together with or without control lentiviral vector or vectors expressing WT or mutant GA T AD2A (7R / K), followed by cell proliferation assay ( ± s.e.m., *** P < 0.001).( K ) MCF7 cells infected with control shRNA (shCTL) or shRNA specific against CARM1 or GA T AD2A, were injected subcutaneously into female B ALB / C nude mice and br ushed with estrogen (E 2 , 10 −2 M) on the neck e v ery tw o da y s f or six w eeks.Mice w ere then euthaniz ed and tumors w ere e x cised and w eighted ( ± s.e.m., * P < 0.05, *** P < 0.001).( L ) MCF7 cells as described in (E) were injected subcutaneously into female BALB / C nude mice and brushed with estrogen (E 2 , 10 −2 M) on the neck e v ery tw o da y s f or six w eeks.Mice w ere then euthaniz ed and tumors w ere e x cised and w eighted ( ± s.e.m., * P < 0.05, N.S., not significant).

Figure 7 .
Figure 7. Targeting CARM1 with EZM2302 inhibits breast cancer cell growth both in vitro and in vivo .( A ) MCF7 cells were transfected with or without Flag-tagged GA T AD2A and treated with or without EZM2302 (25 μM, 48 h), f ollo w ed b y immunoprecipitation (IP) with anti-Flag antibody and immunoblotting (IB) analysis with antibodies as indicated.( B ) MCF7 cells treated with or without EZM2302 (25 μM, 48 h) were subjected to ChIP with control IgG or anti-GA T AD2A specific antibody followed by qPCR analysis with primers specifically targeting promoter regions of selected cell cycle genes as indicated ( ± s.e.m., * P < 0.05, *** P < 0.001).( C ) MCF7 cells treated with or without EZM2302 (25 μM, 48 h) were subjected to RNA extraction and RT-qPCR analysis to examine the expression of selected cell cycle genes as indicated ( ± s.e.m., * P < 0.05, ** P < 0.01, *** P < 0.001).( D-F ) MCF7 cells treated with or without EZM2302 at concentration as indicated were subjected to cell proliferation assay (D), FACS analysis (E), and colon y f ormation assa y (F) ( ± s.e.m., ** P < 0.0 1, *** P < 0.00 1).( G ) Quantification of the cry stal violet dy e as sho wn in (F) ( ± s.e.m., *** P < 0.001).( H ) MCF7 cells were injected subcutaneously into female BALB / C nude mice, and brushed with estrogen (E 2 , 10 −2 M) on the neck e v ery tw o da y s until tumor size reached approximately 100 mm 3 .Mice were then randomly assigned into three groups and treated with or without EZM2302 intraperitoneally e v ery tw o da y s f or 13 da y s.Tumors w ere harv ested, photographed, and w eighted.( I ) T he w eight of tumors in (H) is sho wn ( ± s.e.m., ** P < 0.01, *** P < 0.001).( J ) The growth curve of tumors in (H) is shown.( K ) Tumors as described in (H) were subjected to RNA extraction and R T-qPCR analy sis to e xamine the e xpression of selected cell cy cle genes as indicated ( ± s.e.m., * P < 0.05, ** P < 0.0 1, *** P < 0.00 1).

Figure 8 .
Figure 8.A proposed model of CARM1-mediated NuRD methylation in gene transcriptional regulation and cell cycle control.CARM1 and NuRD commonly bind and activate a large cohort of genes with implications in cell cycle control to facilitate G1 / S transition.Activation of this gene program requires CARM1 to methylate a key subunit in NuRD, GA T AD2A / 2B.Aberrant expression of CARM1 and NuRD results in uncontrolled expression of these cell cycle genes and cell cycle progression, leading to cancers, such as breast cancer.